The present invention relates to a method for detecting whether base sequences of interest are present in a sample DNA or RNA utilizing a bonding of a sample DNA or RNA with probe DNAs or RNAs by means of hybridization. The case wherein a sample or probes are DNA can be considered in a similar way to the case wherein a sample or probes are RNA. Accordingly, the case wherein both of a sample and probes are DNA is described hereinafter as an example.
A conventional means for detecting whether a base sequence of interest is present in a sample DNA is carried out as follows: DNA having a base sequence complementary to a sample DNA of interest to be detected is prepared as a probe DNA and made into a DNA chip by immobilizing it on glass or membrane. The sample DNA is hybridized with the probe DNA immobilized on a DNA chip. When the intended sequence is present in the sample DNA, a site having the base sequence of interest in the sample DNA is bonded to the probe DNA immobilized on the DNA chip to form a hybridization complex. When there is no base sequence of interest in sample DNA, there is no bonding of sample DNA to the probe DNA and, therefore, sample DNA remains in a solution. When the DNA chip is washed after the hybridization, the DNA in solution is removed while bonded sample DNA is not removed. A method for the detection of a probe DNA forming a hybridization complex is effected as follows: The sample DNA which has been previously labeled by a fluorescent substance is radiated by exciting with a light source such as lamp or laser. The formed image is read by an DNA chip reading device to determine whether the base sequence of interest is present in a sample DNA which is hybridized with probe (Science, 270:467-470).
FIG. 2 is an example of a method for the detection of an base sequence of interest using a DNA chip which has been often utilized recently. This example shows a means for the detection whether four different base sequences of interest, i.e. a base sequence of interest (1) 1, a base sequence of interest (2) 2, a base sequence of interest (3) 3 and a base sequence of interest (4) 4 are present in a sample DNA 5. In this example, the base sequence of interest (1) 1 and the base sequence of interest (4) 4 are present in a sample DNA.
FIG. 2(A) is a sample DNA 5 which is previously subjected to a fluorescence labeling and in which the base sequence of interest (1) 1 and the base sequence of interest (4) 4 are present.
FIG. 2(B) is a DNA group 14 comprising DNA 1a of the base sequence of interest (1) and DNA 1b complementary to DNA 1a, DNA 2a of the base sequence of interest (2) and DNA 2b complementary to DNA 2a, DNA 3a of the base sequence of interest (3) and DNA 3b complementary to DNA 3a and DNA 4a of the base sequence of interest (4) and DNA 4b complementary to DNA 4a.
In FIG. 2(C), complementary chain DNAs 1b to 4b from FIG. 2(B) are immobilized on glass or membrane as a probe DNA group 8 to prepare a DNA chip 9 of FIG. 2(D).
In FIG. 2(E), a sample DNA 5 and a probe DNA group 8 immobilized on the DNA chip 9 are placed into a hybridization solution 10 for hybridization. The site of the base sequence of interest (1) 1 in a sample DNA 5 and the DNA 1b of the base sequence of interest (1) are in a relation of complementary chain and, therefore, the site of the base sequence of interest (1) 1 in the sample DNA 5 and the complementary chain DNA 1b are hybridized and bonded.
FIG. 2(F) shows a reading means of DNA chip 9 after the hybridization in which the fluorescence label 6 is emitted by exciting with a lamp 11 and read by a DNA chip reading device 7. In this example, the position of the complementary DNA 1b of the base sequence of interest (1) 1 bonded by means of hybridization complex formation to the site of the base sequence of interest (1) 1 in the sample DNA 5 shines.
FIG. 2(G) is an image 12 read by the DNA chip reading device 7 and, from the shining position, it is found that the base sequence of interest (1) 1 is present in the sample DNA 5. At present, it is detected whether the base sequence of interest is present in a sample DNA by such means.
According to the conventional method, when the amount of a sample DNA is small, only a hybridization of one sample DNA with one probe DNA is possible since the probe DNA is immobilized on glass or membrane. Therefore, there is a problem that, when there are plural kinds of base sequences of interest to be detected in a sample DNA, only one of them is bonded to sample DNA while other base sequences of interest cannot be detected. Accordingly, a sample DNA must be amplified and increased. However, there is a problem that, when the sample DNA is an unknown DNA, sizes of the molecule are various, etc., the condition for the amplification is difficult. In the case of hybridization using a DNA chip, the space where the hybridization is carried out is too large and, therefore, there is a problem that probability to encounter the immobilized probe DNA and a sample DNA is low. In addition, molecular weight of the sample DNA is greatly different from that of the probe DNA and, therefore, there is a problem that it is difficult to set up experimental conditions such as temperature which is a condition for the hybridization.
The present invention has been carried out in view of the problems in the prior art as such. It is an object of the present invention to make hybridization of plural probe DNAs or RNAs with a sample DNA or RNA possible even when the amount of sample DNA or RNA is little and to provide a method where, even when plural kinds of base sequences of interest are present in a sample DNA or RNA, they can be easily detected.